Statistics from Altmetric.com
Cerebral arteriovenous malformations (AVMs) are thought to be congenital lesions exhibiting features of either mature vascular walls or embryonal anastomotic plexuses. It is generally assumed that changes in size are dependent on enlargement of the venous compartment, organisation in the setting of microhaemorrhages, and gliosis. However, recent findings are consistent with the hypothesis of ongoing angiogenesis.1 2
Previous research from this laboratory disclosed that peculiar isoforms of fibronectin (FN) and tenascin (TN) typically occur in fetal and neoplastic tissues.3-5 These isoforms are a blend of structurally different glycoproteins that result from alternative splicing of the primary transcript and are mainly expressed in the extracellular matrix. Their expression is undetectable in normal adult tissues, with the exception of the vessels in the regenerating endometrium. To gain further insight into the pathobiology of the AVMs the present report sought to ascertain whether these lesions also express oncofetal FN and TN isoforms.
Tissue samples were obtained after neurosurgical excisions of ruptured AVMs. All 10 patients had experienced an intracerebral haemorrhage as the first clinical manifestation of their disease. There was no drug history before bleeding. Control specimens from two right gyri recti and one cerebellar tonsil were obtained, respectively, from operations for ruptured aneurysms of the anterior communicating artery or for Arnold Chiari disease.
Immunohistochemical evaluations were performed on 5 μm thick cryostat sections using a protocol reported previously.5 Owing to the limited amount of available material, only in a few cases was some fresh tissue retained to allow western blots. Distribution of FN and TN isoforms was investigated using three monoclonal antibodies (mAbs) or two Ab fragments, obtained by phage display technology, respectively. These Abs, prepared in our laboratory, were found to work on fresh frozen material. According to the previous characterisations the BC-1 mAb and the TN-11 Ab fragments are specific for isoforms occurring almost exclusively in fetal tissues and in tumours, with the recognised TN isoform being typically associated with anaplastic gliomas (table).
Control sections were processed identically to the other specimens, but the primary antibody was substituted with a specific immunoglobulin of recombinant antibodies. The antibodies were blocked using the specific antigens. The antigens were recombinant protein containing the epitope produced in E Coli. For the mAb BC-1 we used the recombinant protein containing the type-III repeats 7B-8–9. For the mAb IST-4 we used the recombinant protein containing the type-III repeats 2–8. For the recombinant antibodies TN-11 and TN-12 the recombinant type-III repeat C and the recombinant fragment containing the EGF-like repeats were used, respectively.
All 10 AVMs were found to contain large amounts of FN and TN, as shown by intense immunostaining with the use of the IST-9 / IST-4 mAbs and the TN-12 Ab fragment. The staining was localised either in the endothelium or the subendothelial layer. A positive response was found in several artery-like vessels and in a few vessels with thinner walls using the mAb BC-1. Staining with the TN-11 Ab fragment showed occurrence of type III repeat C TN isoform in the inner layers of the vascular components of the nidus, irrespective of their morphology.
Six out of the 10 examined specimens were found to contain portions of cerebral tissue surrounding the angiomatous nidus. In all these cases the wall of several vessels exhibited intense staining with the use of the TN-11 Ab fragment. Using the BC-1 mAb some of these vessels exhibited some staining (figure). In the control specimens (brain and cerebellum) both the FN isoform containing the ED-B sequence (ED-B+FN), and the type III repeat C TN isoform were absent, despite the widespread distribution of total FN and TN in the vascular walls.
Previous findings showed that ED-B+FN presents with conformational modifications in its central part and results from deregulation of FN pre-mRNA.4 The distribution of this isoform was found to be highly restricted in normal adult tissues. By contrast, ED-B+ FN exhibited widespread distribution in the vasculature of fetal tissues, including brain, and of several types of malignancies. It was therefore regarded as a marker of angiogenesis.5
Similarly, the type III repeat C TN isoform, recognised by the Ab fragment TN-11, was found to occur in the vascular walls of anaplastic gliomas. Northern blot analysis showed that the mRNA of this isoform was undetectable in normal tissues and some malignancies, but was present in large amounts in fetal tissues, including brain, and in glioblastomas3
Recent advances in the pathology of cerebral AVMs suggest that these lesions might not be static. Tyrosine kinase, an endothelial cell specific receptor upregulated in glioblastomas, was found to be highly expressed in both AVMs and in the vessels of cerebral tissue bordering the malformations, by contrast with the down regulation occurring in the vasculature of the normal brain.1The pattern of distribution of structural proteins was consistent with the hypothesis of diffuse activation of angiogenesis, without specific relation to individual vessel types.2
Furthermore, use of the cell proliferation marker MIB-1 showed endothelial proliferation in arterioles, venules, and capillaries of the cerebral tissue neighbouring AVMs.1
The present findings indicate that a particular FN isoform, mainly expressed by the vasculature of fetal and tumorous tissues, as well as a TN isoform typically detected in the walls of vessels in anaplastic gliomas, also occur in AVMs and in vessels of adjacent cerebral tissue, but that both isoforms are absent in normal brain. This evidence provides further support to the hypothesis of ongoing angiogenesis in and around these lesions.
The presence of angiogenic features in AVMs might result from maintenance of proliferating and remodelling potentials, or from a specific response to haemodynamic stress in vascular structures subjected to increased blood flow and pressure. Occurrence of these features also in vessels lying in areas peripheral to the nidus might be related to recruitment of the neighbouring vasculature, possibly dependent on focal ischaemia in the setting of arteriovenous shunting.1 2 However, the presence in apparently normal vasculature of molecules typically occurring in fetal tissues and malignancies indicate that cerebral AVMs may not be static lesions. Further studies are needed to ascertain whether this phenomenon results merely from haemodynamic stress or actually reflects an intrinsic growth potential. Should this second be the case, current therapeutic strategies would possibly require revision.
This study was partially supported by the National Research Council (CNR), AIRC and the Ministry of University and Scientific Research (MURST). We thank Sergio Deseri, EE, for his technical help and Mr. Thomas Wiley for manuscript revision.
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.